**Background**
Proteolytic cleavage is a fundamental biological process essential for the activation of various zymogens and the processing of fusion proteins in biotechnology. Among these, enterokinase (EK) serves as a critical serine protease that functions as the physiological activator of trypsinogen, converting it into its active form, trypsin. This conversion is a pivotal step in the digestive cascade within the small intestine. In laboratory settings, the high specificity of enterokinase for the sequence Asp-Asp-Asp-Asp-Lys makes it an indispensable tool for the precise cleavage of recombinant proteins and the removal of fusion tags. Given its importance in protein engineering and physiological studies, researchers require high-activity enzymes to ensure efficient processing. In this context, we will introduce a high-activity serine protease – Recombinant enterokinase.
**Definition**
Recombinant enterokinase is a recombinant serine protease with an enzyme activity of ≥5 U/μL. One unit is defined as the amount of enzyme required for 95% cleavage of 0.5 mg of fusion protein stored in 25 mM Tris-HCl 8.0 buffer at 25°C for 12 to 16 hours.
**In Vitro Studies**
The Recombinant enterokinase description highlights its role in the precise activation of trypsinogen and the cleavage of fusion proteins. According to the Recombinant enterokinase protocol, the enzyme can be diluted with 25 mM Tris-HCl pH 8.0 to a working solution of 0.1 U per 1 μL. For enzyme digestion, a reaction system typically contains 0.05-0.1 mg of fusion protein (at a concentration of 0.1-1 mg/mL) and 0.1-0.2 U of the enzyme. The digestion is performed at 25°C overnight, with complete cleavage generally requiring 16-24 hours.
Recombinant enterokinase in vitro performance is highly sensitive to reaction conditions. It is recommended to avoid temperatures of 37°C to prevent non-specific digestion. Furthermore, the enzyme’s activity is inhibited by phosphate and high concentrations of imidazole (>200 mM), NaCl (>200 mM), or glycerol (>5%). To achieve ideal results, samples should be dialyzed into 25 mM Tris-HCl 8.0 buffer. If the protease-cutting effect is unsatisfactory, the enzyme dosage or incubation time can be increased. For downstream applications requiring the removal of the enzyme, anion exchange resins such as DEAE-FF can be used with an elution buffer of 25 mM Tris-HCl pH 8.0 containing 100 mM NaCl. In conclusion, Recombinant enterokinase is a highly efficient and specific serine protease suitable for the activation of trypsinogen and the cleavage of fusion proteins.
Keywords
Recombinant enterokinase, 9014-74-8, rEK, Ser/Thr Protease, Serine proteases, Serine endopeptidases, Threonine proteases, Inhibitor, inhibitor, inhibit
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